Method for chromosomal rearrangement by consecutive gene targeting of two recombination substrates to the deletion endpoints

ABSTRACT

The present invention involves the creation of defined chromosomal deficiencies, inversions and duplications using Cre recombinase in ES cells transmitted into the mouse germ line. These chromosomal reconstructions can extend up to 3-4 cM. Chromosomal rearrangements are the major cause of inherited human disease and fetal loss. Additionally, translocations and deletions are recognized as major genetic changes that are causally involved in neoplasia. Chromosomal variants such as deletions and inversions are exploited commonly as genetic tools in organisms such as Drosophila. Mice with defined regions of segmental haploidy are useful for genetic screening and allow accurate models of human chromosomal disease to be generated.

[0001] The present invention was made utilizing funds of the UnitedStates Government. The U.S. Government is entitled to certain rightsunder this invention.

BACKGROUND OF THE INVENTION

[0002] 1. Technical Field of the Invention

[0003] The present invention involves the creation of definedchromosomal deficiencies, inversions and duplications using Crerecombinase in embryonic stem cells and transmitted into the mouse germline. In the present invention, these chromosomal reconstructions canextend up to 3-4 cM. Chromosomal rearrangements are the major cause ofinherited human disease and fetal loss. Further, chromosomaltranslocations and deletions are recognized as major genetic changesthat are causally involved in neoplasia. Chromosomal variants such asdeletions and inversions are exploited commonly as genetic tools indiploid organisms such as Drosophila. In diploid organisms, suchdeficiencies are exploited in genetic screens because a small portion ofthe genome is functionally hemizygous. Thus, a mutation which wouldnormally be recessive and masked by the wildtype allele in a diploidcontext will be dominant and detectable in the haploid state. In themouse, deficiencies have not, up to now, been available generally; thus,screens for recessive mutations are nonexistent or particularlycumbersome. However, the present invention provides methods to engineermice and cell lines with defined regions of segmental haploidy. Suchmice are useful for genetic screening and provide accurate models ofhuman chromosomal diseases.

[0004] 2. The Prior Art

[0005] Inherited chromosomal rearrangements such as inversions,duplications and deficiencies are responsible for a significant fractionof human congenital disease. Chromosomal changes also occur somaticallyand are associated with neoplastic disease. Defining the causal geneticalteration in a region of the genome associated with chromosomalrearrangements can be relatively straightforward if the affected genelies in the breakpoint of an inversion or translocation. However, incases of duplications and deficiencies, the specific genetic lesion(s)associated with pathological chromosomal changes are much harder toidentify. Still, the generation of animal models that accuratelyrecapitulate the genetic lesion would facilitate the study of diseaseand could be very helpful in the efforts to dissect specificgene-function relationships in multigene syndromes.

[0006] In diploid organisms such as Drosophila, chromosomal deficienciesare commonly exploited in genetic screens because a small portion of thegenome is functionally hemizygous. Thus, a mutation which would berecessive and masked by the wildtype allele in the diploid context willbe dominant and therefore readily detectable in the haploid state. Inthe mouse, deficiencies are not available generally. Despite the limitednumber of deficiencies available in the mouse, the potential for thedetailed analysis of a genetic interval using these deficiencies hasbeen demonstrated clearly. See Holdener-Kenny, et al., BioEssays,14:831-39 (1992), which is hereby incorporated by reference.

[0007] Deficiencies that are available currently in the mouse genomewere generated at random using ionizing irradiation. Althoughconventional gene targeting technology in embryonic stem (ES) cells cangenerate virtually any type of mutation, including deletions of up to 20kb, it has not been possible to delete substantially larger fragments byusing standard methodology. Likewise, the technology required toconstruct large inversions and duplications has not been established.

[0008] One mechanism by which chromosomes may be engineered is by theuse of Cre recombinase. Cre recombinase has been used in mammalian celllines and in vivo to delete or invert sequences between the 34 base pairrecognition sequences, loxP sites, placed a few kb apart on the samechromosome. The recombination is initiated by Cre proteins which bind to13-bp inverted regions in the loxP sites and promote synapses or joiningof a pair of sites. Next, the Cre proteins catalyze strand exchangebetween the pair of sites within an asymmetric 8-bp central spacersequence by concerted cleavage and rejoining reactions, involving atransient DNA-protein covalent linkage. Smith, et al., Nature Genetics,9:376-385 (1995); Gu, et al., Science, 265:103-06 (1994) and Sauer,Nucl. Acids Res., 17:147-61 (1989) (both of these references are herebyincorporated by reference). Additionally, recombinases have been used toinduce mitotic recombination between homologous and non-homologouschromosomes in Drosophila, plants and mammalian cells. Embryonic stemcell technology has become a powerful tool for defining the function ofmammalian genes, but mainly has been restricted to the mutation ofsingle genes. Replacement vectors have been used to construct deletionsof up to 19 kb; however, utilizing the same strategy to construct largerdeletions (>60 kb) has not been successful. In the present invention,the generation and direct selection of deletions, duplications andinversions, ranging from 90 kb to 3-4 cM, in ES cells is demonstrated.

SUMMARY OF THE INVENTION

[0009] The method of the present invention is based on consecutive genetargeting of two recombination substrates to the deletion endpoints andthe subsequent induction of recombination mediated by the Crerecombinase. This method generates a positive selectable marker allowingfor the direct selection of clones with the desired chromosomestructures. Despite the multitude of steps involved in generating theserearrangements in ES cells, deletion and duplication alleles have beentransmitted into the mouse genome.

[0010] One object of the present invention is a method for causing alarge-scale chromosomal rearrangement by first deleting a portion ofgenetic material.

[0011] An additional object of the present invention is a targetingvector system capable of inserting into two endpoint regionsconstraining a desired chromosomal deletion.

[0012] Thus in accomplishing the foregoing objects, there is provided inaccordance with one aspect of the present invention a method fordeleting a selected region of genetic material in cells comprising thesteps of: inserting a first selection cassette at a 5′ end of saidselected region using conventional gene targeting methods, said firstselection cassette comprising a first selectable marker, a first loxPrecombination site, and a first portion of a second selectable marker;selecting cells expressing said first selectable marker; inserting asecond selection cassette at a 3′ end of said selected region usingconventional gene targeting methods, said second selection cassettecomprising a third selectable marker, a second loxP recombination site,and a remaining portion of said second selectable marker; selectingcells expressing said third selectable marker; expressing Crerecombinase to produce recombination between said first and second loxPsites; and selecting cells expressing said second selectable marker.

[0013] Specific embodiments of the above method can include a puromycinresistance gene as the first selectable marker, a functional Hprt geneas the second selectable marker, and a neomycin resistance gene as thethird selectable marker. Numerous other selectable markers will work,their presence in the particular deletion strategy is merely to aid cellselection. In other preferred embodiments, the first selectable markeris a puromycin resistance gene. In still other preferred embodiments,the second selectable marker is a functional Hprt gene. And in stillother preferred embodiments, the third selectable marker is a neomycinresistance gene.

[0014] In still other preferred embodiments, the cells referred to aboveare embryonic stem cells, though significant, they need not be stemcells. In other preferred embodiments, the cells are embryonic stemcells, and said cells develop into mice. And in yet other preferredembodiments, the cells are embryonic stem cells, and said cells aremaintained as cell lines.

[0015] In yet another preferred embodiment, a viral vector is used toreplace either or both native sequences of DNA. In one embodiment, thisvirus is a retrovirus. In yet another embodiment, the viral vectorreferred to above has a provirus structure comprising a cassette in turncomprising: an hprtΔ5′ cassette, a loxP site, and a puromycin resistancegene.

[0016] In yet another particularly preferred embodiment, the method fordeleting a portion of chromosomal material in cells wherein thetargeting vectors are a first targeting vector for replacing said firstnative sequence of DNA at said 5′ end, comprising: a genomic insertcloned into the vector of about 7.5 kb; a tyrosinase minigene; a Neo^(r)gene; a 5′ hprt fragment; and a loxP site embedded into said hprtfragment; and a second targeting vector for replacing said second nativesequence of DNA at said 3′ end, comprising: a genomic insert cloned intothe vector of about 8.5 kb; a K14-Agouti gene; a Puro^(r) gene; a 3′hprt fragment; and a loxP site embedded into said hprt fragment.

[0017] In one particularly preferred embodiment of the second aspect ofthe present invention, there is provided a replacement vector systemcomprising a first targeting vector for replacing said first nativesequence of DNA at said 5′ end, comprising a genomic insert cloned intothe vector of about 7.5 kb; a tyrosinase minigene; a Neo^(r) gene; a 5′hprt fragment; and a loxP site embedded into said hprt fragment; and asecond targeting vector for replacing said second native sequence of DNAat said 3′ end, comprising: a genomic insert cloned into the vector ofabout 8.5 kb; a K14-Agouti gene; a Puro^(r) gene; a 3′ hprt fragment;and a loxP site embedded into said hprt fragment.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1: 1A: depicts the Hprt-loxP minigene cassette; 1B: depictsthe hprtΔ5′ and hprtΔ3′ recombination substrates; 1C: outlines thegeneral strategy for Cre-induced, targeted genomic rearrangementsillustrated at the HoxB cluster—only the intrachromosomal pathway isshown; 1D: demonstrates alternative orientations (A or B) of therecombination substrates at the E2DH and Gastrin loci; 1E: showschromosomal alterations induced by Cre recombinase for the differentorientations of the minigenes in cis and trans.

[0019]FIG. 2: Shows the results of a Southern blot analysis of thechromosomal engineering technology used to delete the HoxB cluster. A-Cdemonstrates the interpretation of, and D-F shows the actual Southernblot data from wildtype (wt), double targeted (dt) or HAT resistant EScell clones (lanes 1 and 2). M1 and M2 are HindIII-cut and BstEII-cutlambda DNA molecular weight markers, respectively. Hoxb-1 is located ina 7.2 kb NheI fragment detected with probe a (2A and 2D; this allele ispresent in all of the lanes). Targeting of the hprtΔ3′-neo cassette tothe Hoxb-1 locus generates a novel 10.2 kb NheI restriction fragmentdetected with probe a (2B and 2D dt). Hoxb-9 is located on a 16 kb NheIrestriction fragment detected with probe b (2A and 2E; this allele ispresent in all of the lanes). Targeting of the hprtΔ5′-puro to theHoxb-9 gene generates a novel 20.4 kb NheI restriction fragment detectedwith probe b (2B and 2E dt). Cre-induced recombination brings togetherthe hprtΔ5′ and hprtΔ3 ′ and produces a NheI 18.2 kb deletion-specificjunction fragment detected by both probes a and b (2C, 2D 1 and 2).Probe c, located in the deletion region, shows a dosage difference inPanel 2F, 1 and 2, compared to the wt and dt lanes. Probe a is a 0.7 kbRsaI fragment located approximately 3 kb downstream of Hoxb-1 exon 2;probe b is a 1 kb RsaI fragment located approximately 5 kb upstream ofHoxb-1 exon 1. P and N represent the puromycin and neomycin selectioncassettes.

[0020]FIG. 3: 3A: depicts mouse chromosome 11, and the loci used asendpoints for the chromosome engineering are illustrated. 3B: shows NheIrestriction sites (N) and fragment lengths around the E2DH and Gastrinloci. 3C: shows chromosome 11 with both the E2DH and Gastrin locitargeted with the hprtΔ5′ and hprtΔ3′ vectors, respectively. Forclarity, only the cis configuration and the A orientation are shown. 3D:shows the structure of the deletion, duplication and inversion alleles.The sizes of the diagnostic restriction fragments and probes used todetect these alleles are indicated. The deletion, duplication andinversion alleles are derived from the double targeted chromosome in theAA, BB and AB configurations, respectively. 3E: shows the results ofSouthern blots which confirm the structure of the recombinantchromosomes. The probes used with each blot (a, b, c or d) are indicatedbeneath each panel and on the diagrams of the various alleles. The lanesare coded as follows: wildtype (wt), double targeted (dt), deletion(del), duplication (dup) and inversion (inv).

[0021]FIG. 4: G₂ trans recombination between homologous chromosomes. 4A:depicts individual sister chromatids from chromosome homologues stilljoined at the centromere. One chromosome is illustrated with hprtΔ5′,the neomycin resistance gene (N) used for targeting. The other homologuewas targeted with hprtΔ3′ cassette linked to the puromycin resistancegene (P). Cre-induced recombination between loxP sites on sisterchromatids from different homologues is illustrated by an X. B:illustrates the recombinant structure of the sister chromatids. Theindividual chromatids are numbered 1-4. Chromatid 3 carries thereconstructed Hprt minigene and the deletion. 4C: shows the results ofHAT selection for chromatid 3 which will segregate with either chromatid1 or 2 which carries only the neo or both the neo and puro cassettes,respectively. The chromatid 2+3 segregant carries a duplication anddeletion (genetically balanced) and is indistinguishable from the G₁inter-chromosomal product. The chromatid 1+3 product carries thedeletion and the original single targeted chromosome; this can only havearisen via the G₂ pathway.

[0022]FIG. 5: Gene dosage analysis and segregation of the deletion andduplication chromosomes through the mouse germ line are demonstrated:Lane 1: wildtype allele (AB2.2 ES cell line); Lane 2: ES cell clone withthe duplication and deletion (genetically balanced); Lanes 3 to 6:transmission and segregation of the duplication and deletion alleles inthe progeny of a chimeric male constructed from the cells shown in lane2. Lanes 3 and 4 show mice with the deletion, lanes 5 and 6 show micewith the duplication; the increase or decrease in intensity of the 9.0kb fragment relative to the 2.0 kb control fragment is consistent withjunction fragment analysis of the inheritance of the duplication ordeletion alleles from these mice (data not shown). Lanes 7 and 8 showmice from heterozygous mating homozygous for the duplication allelewhich is evident from the increased intensity of the 9.0 kb fragment.

[0023]FIG. 6: Deletion of two 3-4 cM intervals on mouse chromosome 11 isshown. 6A depicts mouse chromosome 11. The shaded bars indicate theintervals which are to be deleted. Hox B, E2DH and Wnt3 are the lociwhich serve as the deletion endpoints. SBC (Sporadic Breast Cancer) lociare indicated by the black bars. These loci are the putative location oftumor suppressor genes based on the analysis of loss of heterozygosityin breast cancer. 6B depicts a double-targeted chromosome which has beentargeted with the hprtΔ3′ cassettes to the Hoxb9 and E2DH genes or tothe E2DH and Wnt3 genes. The A orientation E2DH-targeted clones wereused with the HoxB-E2DH deletion and the B orientation clones were usedwith the E2DH-Wnt3 deletion. Only the orientations of the hprtΔ5′cassette which give the deletion products are illustrated. The verticalbars represent NheI sites; the sizes of the fragments are indicated andthe probes are indicated by shaded boxes labelled c and d. 6C shows thestructure of deletion alleles. Diagnostic NheI fragments for thedeletion are indicated. 6D reveals the Southern analysis that confirmsthe structure of the alleles in the wildtype (wt), double-targeted (dt)and deletion (dt) clones. The deletion-specific junction fragments areindicated by the arrows.

[0024]FIG. 7: Schematic representation of a provirus structure,containing hprtΔ5′ minigene cassette, a loxP site, and a puromycinresistance gene, for use as a vector in one embodiment of the presentinvention. This particular vector would insert the loxP site at the 5′end of the chromosome.

[0025]FIG. 8: A 3′ anchor library that contains the expression cassette3′ hprt, puromycin resistance gene, and k14-agouti gene.

[0026]FIG. 9: A 5′ anchor library that contains the expression cassettes5′ hprt, neomycin resistance gene, and tyrosinase gene.

[0027]FIG. 10: Map of an exemplary 5′ endpoint targeting vectorautomatically excised out of a phage clone isolated from the 5′ anchorlibrary.

[0028]FIG. 11: Map of an exemplary 3′ endpoint targeting vectorautomatically excised out of a phage clone isolated from the 3′ anchorlibrary.

[0029]FIG. 12: Map of pG12WT (Wildtype 3′ hprt cassette plasmid formaking chromosomal rearrangements). The sequence is identical to pG12except that the mutation in 3′ hprt has been fixed.

[0030]FIG. 13: Map of a portion of the mouse chromosome 11 showing thegeneral composition of the selection cassettes positioned at thechromosome endpoints, and the position of the Ore-induced deletioninterval, E₂DH-D11Mit199.

[0031]FIG. 14: Map of a portion of the mouse chromosome 11 showing thegeneral composition of the selection cassettes positioned at thechromosome endpoints, and the position of the Cre-induced deletioninterval, E₂DH-D11Mit69.

DETAILED DESCRIPTION OF THE INVENTION

[0032] It will be apparent readily to one skilled in the art thatvarious substitutions and modifications may be made to the inventiondisclosed herein without departing from the scope and spirit of theinvention.

[0033] As used herein, the term “chromosome engineering” means creatingchromosome inversions, duplications or deletions.

[0034] As used herein, the term “chromosome deficiency” means a lack ofa chromosome or portion of a chromosome.

[0035] As used herein, the term “chromosome inversion” means reversal ofa part of a chromosome so that the genes within that part are in reverseorder.

[0036] As used herein, the term “chromosome duplication” means an extra,duplicate chromosome or part of a chromosome.

[0037] As used herein, the term “ES cell” stands for embryonic stemcells: cells which are derived from early mouse embryos that can bemaintained in an undifferentiated state, but, upon return to theenvironment of the early embryo, can contribute to all types of cells inthe resulting chimera.

[0038] As used herein, the term “Cre-induced recombination” meanscatalysis of both intramolecular and intermolecular recombination by theCre protein. The Cre protein is a 38 kD protein that recombines DNAbetween specific, 34 bp sequences called loxP sites.

[0039] As used herein, the term “interchromosomal recombination” meansrecombination between different chromosomes.

[0040] As used herein, the term “intrachromosomal recombination” meansrecombination between regions on the same chromosome.

[0041] As used herein, the term “HoxB” refers to a specific gene clusterhaving the same physical distance as a P1 phage and having knownstructure and orientation.

[0042] As used herein, the term “Hoxb-9” refers to a specific gene inthe HoxB gene cluster.

[0043] As used herein, the term “Hoxb-1” means the 3′-most gene of theHoxB gene cluster.

[0044] As used herein, the term “hprt” means hypoxanthinephosphoribosyltransferase.

[0045] As used herein, the term “loxP site” means the specific 34 bpsequence recognized by Cre recombinase.

[0046] As used herein, the term “G418 resistance” means having a genewhich confers resistance to G418.

[0047] As used herein, the term “neo resistance” means having a genewhich confers resistance to G418.

[0048] As used herein, the term “puromycin resistance” means having agene which confers resistance to puromycin.

[0049] As used herein, the term “HAT resistance” means cells resistantto media containing hypoxanthine, aminopterin and thymine. Only cellsexpressing the Hypoxanthine phosphoribosyl transferase (Hprt) andthymidine kinase genes will grow in HAT medium.

[0050] As used herein, the term “targeting deletion” means a planneddeletion created by targeting an area for recombination by insertion ofa loxP or other recombination site.

[0051] As used herein, the term “germ line transmission” refers to achimeric animal capable of transmitting a particular trait to itsoffspring.

[0052] As used herein, the term “hemizygous” means genes present onlyonce in a genotype.

[0053] As used herein, the term “heterozygous” refers to the state of anorganism having two different alleles at a given locus on homologouschromosomes.

[0054] As used herein, the term “homozygous” refers to the state of anorganism having the same two alleles at a given locus on homologouschromosomes.

[0055] As used herein, the term “Gastrin” locus means a gene on mousechromosome 11 which encodes a peptide involved in stimulating acidsecretion in the stomach. See Fuller et al., Molec Endocrinol. 1:306-11(1987).

[0056] As used herein, the term “E2DH” locus, also known as 17HSD, is agene whose product is involved in steroid biosynthesis. Thisdehydrogenase converts estrone to estradiol. See The et al., Molec.Endocrinol. 3:1301-06 (1989).

[0057] As used herein, the term “Wnt3” locus is a gene which encodes amember of the wnt family of growth factors. Wnt3 is a target foractivation by MMTV which causes mammary tumors. Roelink et al., PNAS87:4519 (1990).

[0058] As used herein, the term “selected region” refers to thatparticular region of the chromosome targeted for manipulation (i.e.,deletion, inversion, duplication).

[0059] In one embodiment of the present invention, a method is disclosedand claimed for deleting a selected region of genetic material in cellscomprising the steps of: inserting a first selection cassette at a 5′end of said selected region using conventional gene targeting methods,said first selection cassette comprising a first selectable marker, afirst loxP recombination site, and a first portion of a secondselectable marker; selecting cells expressing said first selectablemarker; inserting a second selection cassette at a 3′ end of saidselected region using conventional gene targeting methods, said secondselection cassette comprising a third selectable marker, a second loxPrecombination site, and a remaining portion of said second selectablemarker; selecting cells expressing said third selectable marker;expressing Cre recombinase to produce recombination between said firstand second loxP sites; and selecting cells expressing said secondselectable marker.

[0060] In particularly preferred embodiments of the present invention,the method referred to above uses a first selectable marker, a puromycinresistance gene, said second selectable marker is an Hprt gene, and saidthird selectable marker is a neomycin resistance gene.

[0061] In another particularly preferred embodiment, the firstselectable marker is a puromycin resistance gene. In yet anotherparticularly preferred embodiment, the second selectable marker is afunctional Hprt gene. In still another preferred embodiment, the thirdselectable marker is a neomycin resistance gene. In another preferredembodiment, the cells are embryonic stem cells. In still anotherpreferred embodiment, the cells are embryonic stem cells, and said cellsdevelop into mice. In still another preferred embodiment, the cells areembryonic stem cells, and said cells are maintained as cell lines. Inanother embodiment of the present invention, Cre is transientlyexpressed Cre. In other embodiments, it is expressed either inducibly orconstitutively.

[0062] In a second general embodiment of the present invention, a methodis disclosed and claimed for deleting a selected region of geneticmaterial in cells comprising the steps of: inserting a first selectioncassette at a 5′ end of said selected region using either conventionaltargeting methods or a viral vector, said first selection cassettecomprising a first selectable marker, a first loxP recombination site,and a first portion of a second selectable marker; selecting cellsexpressing said first selectable marker; inserting a second selectioncassette at a 3′ end of said selected region using conventional genetargeting methods or a viral vector, said second selection cassettecomprising a third selectable marker, a second loxP recombination site,and a remaining portion of said second selectable marker; selectingcells expressing said third selectable marker; expressing transientlyCre recombinase to produce recombination between said first and secondloxP sites; and selecting cells expressing said second selectablemarker.

[0063] In one particularly preferred embodiment, the viral vector is aretrovirus. In yet another particularly preferred embodiment, the viralvector has a provirus structure comprising a cassette in turn comprisingan hprtΔ5′ cassette, a loxP site, and a puromycin resistance gene. Inyet another particularly preferred embodiment, the viral vector has aprovirus structure comprising a cassette in turn comprising an hprtΔ5′cassette, a loxP site, and a neomycin resistance gene. In still anotherparticularly preferred embodiment, the targeting or viral vectors are afirst vector for inserting said first native sequence of DNA at said 5′end, comprising: a genomic insert cloned into the vector of about 7.5kb; a tyrosinase minigene; a Neo^(r) gene; a 5′ hprt fragment; and aloxP site embedded into said hprt fragment; and a second vector forinserting said second native sequence of DNA at said 3′ end, comprising:a genomic insert cloned into the vector of about 8.5 kb; a K14-Agoutigene; a Puro^(r) gene; a 3′ hprt fragment; and a loxP site embedded intosaid hprt fragment.

[0064] In a third general embodiment of the present invention, areplacement vector system, is disclosed and claimed comprising: a firstvector for inserting said first native sequence of DNA at said 5′ end,comprising: a genomic insert cloned into the vector of about 7.5 kb; atyrosinase minigene; a Neo^(r) gene; a 5′ hprt fragment; and a loxP siteembedded into said hprt fragment; and a second vector for inserting saidsecond native sequence of DNA at said 3′ end, comprising: a genomicinsert cloned into the vector of about 8.5 kb; a K14-Agouti gene; aPuro^(r) gene; a 3′ hprt fragment; and a loxP site embedded into saidhprt fragment.

[0065] In a fourth general embodiment of the present invention, there isdisclosed and claimed a method for creating defined chromosomaldeficiencies, deletions, and duplications comprising the steps of:identifying a desired region of a chromosome of interest to be deleted;inserting two native sequences at each endpoint of said region of saidchromosome of interest using a first and a second targeting vector, eachcomprised of one or more selectable markers and a loxP site and an hprtfragment; transiently expressing Cre recombinase to producerecombination between each of two said loxP sites; whereby uponchromosomal rearrangement induced by said Cre recombinase, a functionalHprt expression cassette is reconstructed.

[0066] Other and further embodiments, features and advantages will beapparent and the invention more readily understood from a reading of thefollowing Examples and by reference to the accompanying drawings forminga part thereof, wherein the examples of the presently preferredembodiments of the invention are given for the purposes of disclosure.

EXAMPLE A General Strategies

[0067] The various chromosomal rearrangements described herein aredesigned with strong positive selection for the desired chromosomalchange. Very generally, this was accomplished by targeting consecutivelycomplementary, overlapping but non-functional hprt-loxP expressioncassettes to the endpoints of a chromosomal interval. Cre expression(either transiently, inducibly, or constitutively) in thesedouble-targeted ES cells induces loxP recombination resulting inchromosomal rearrangements specific to the relative orientation of theloxP sites. Since the loxP sites are imbedded in the hprt minigenefragments, the chromosomal rearrangement will also reconstruct afunctional hprt expression cassette, therefore facilitating directpositive selection for the clones with these alterations.

[0068] The use of mouse ES (embryonic stem) cells is preferable, thoughnot required, to execute the method the present invention. Use of thesecells would, of course, allow large-scale chromosome manipulation to beintroduced into a germ line, which would in turn facilitate enhancedfunctional study of the mouse genome.

[0069] The loxP sites were introduced by conventional gene targetingprotocols or by viral vectors into the endpoints of the region which wasto be rearranged. Or, one endpoint can be introduced by conventionalmethods, and the other introduced by a viral vector. To maximize theability to select for the rare ES cell clones in which Cre expressionhad successfully induced recombination between loxP sites, theindividual loxP sites targeted to the endpoints of the chromosomalrearrangement were imbedded in two complementary but non-functionalfragments of an Hprt minigene cassette. Recombination between the loxPsites would restore the activity of this cassette, facilitating thedirect selection in HAT media of only those recombinant ES cells withthe desired chromosomal structure (FIG. 1A and B).

[0070] In one particularly preferred embodiment of the presentinvention, the complementary recombination/selection substrates consistof overlapping, but incomplete, pieces of an Hprt minigene with a loxPsite in the intron. These minigene fragments are linked to differentpositive selection cassettes which are required for selection duringgene targeting. The 5′ fragment of the loxP-Hprt minigene is linked to aneomycin resistance gene (hprtΔ3′ cassette), while the 3′ fragment islinked to a puromycin resistance gene (hprtΔ5′ cassette). Cre-inducedrecombination between the loxP sites generates a fully-functional Hprtminigene which provides resistance to HAT selection in Hprt-deficientcells. The positive selectable markers are positioned so that followingrecombination, they are lost from the deleted chromosome. All of theclones that survive selection have the desired chromosomal structure. Asimilar positive selection system for detecting a chromosomaltranslocation has recently been reported by Smith et al., NatureGenetics 9:376-385 (1995). In addition, genes such as K14-agouti andtyrosinase genes can be preferably inserted into the vectors for use ascolor-coat markers, to aid in selecting the members of the populationfor which the chromosomal insert was successful. Albino mice lack thetyrosinase gene, so reinsertion of that gene is manifest by black micein a population of white mice. Similarly, the k14-agouti gene givesyellow color to the tips of the coat hairs against a black background(i.e., it makes black mice appear brown).

[0071] Initially, a small deletion (90 kb) was constructed whichencompasses the HoxB locus since the gene order and orientation wasknown. Subsequently, much larger chromosomal alterations were generated.For the latter alterations, knowledge of the transcriptional directionof the genes which serve as the rearrangement endpoints was notavailable. Consequently, it was necessary to generate ES cell lines withthe four possible configurations of the hprt minigene fragments. Becausethe transcriptional direction of the genes relative to the centromerewas also unknown, it was not possible to predict which combination oforientations would give a deletion; however, clones with deletions arereadily distinguished in culture from the clones with other classes ofrecombinant chromosomes because in addition to becoming HAT resistant,both of the positive selection markers are lost. The generation of adeletion reveals the relative transcriptional direction of the twodeletion endpoints, and if the proximal-distal map positions are known(which was the case in these experiments), further deletions from thesame endpoint are greatly simplified.

[0072] The frequency of recombination between the loxP sites when theywere on the same chromosome varied from 6×10⁻⁷ to 5×10⁻⁵, but a directrelationship between the distance and the frequency was not apparent.The frequency of recombination was, however, significantly lower thanthose reported when the loxP sites are a few kb apart; see, Gu, et al.,Cell 73:1155-64 (1993), verifying that selection is required to isolatethese clones. These frequencies are derived by the transienttransfection of Cre in ES cells, but might be higher under conditions ofconstitutive expression of Cre, for example, in a specific lineage in atransgenic mouse. The frequency of recombination was reduced by one totwo orders of magnitude when the loxP sites were integrated in transcompared to the cis configuration. This is consistent with the knowledgethat individual chromosomes occupy discrete, non-overlapping domains inan interphase nucleus.

[0073] HAT-resistant clones derived from the trans configuration of thedouble-targeted clones oriented to give deletion products were notexpected to become G418 or puro sensitive. But approximately half of theHAT-resistant clones segregated the puro cassette while all retained theneo cassette. This segregation pattern is consistent withinter-sister-chromatid recombination (see FIG. 4). Although the numberof clones with the trans configuration was relatively small, the equalratio of puro+neo to neo-only segregants suggests that G₂ recombinationis the predominant pathway used in this case. The rescue of hprtnegative daughter cells by metabolic cooperation also suggests that asubstantial fraction of the HAT-resistant clones derived from the cisdouble-targeted clones may have been generated by the sister-chromatidpathway.

[0074] The correlation of the induced chromosomal rearrangements withthe orientation of the vectors has revealed physical mapping informationin this region of mouse chromosome 11. For instance, the genes describedin the following Examples, Gastrin, E2DH, Wnt 3 and the Hox B cluster,are all transcribed in the centromere-to-telomere direction. TheE2DH-HoxB deletion has shown that the HoxB cluster is oriented with theHoxb-9 gene nearest to the centromere.

EXAMPLE B Deletion and Duplication of 90 kb Containing the HoxB Cluster

[0075] The HoxB cluster provides an excellent substrate for the deletionstrategy since the cluster is about the same physical distance as a P1phage and the structure and orientation of the individual genes isknown. See, Rubock, et al., PNAS USA 87:4751-55 (1990). Moreover, adeletion allele of HoxB is very useful for detailed genetic analysis ofthis region.

[0076] To generate a HoxB deletion allele, the strategy outlined in FIG.1 was followed. The hprtΔ3′ cassette was used to construct a targetingvector for Hoxb-1, the most 3′ gene of the cluster, and targeted cloneswere identified (FIG. 2). An ES clone with the Hoxb-1 targeted allele(FIG. 2B) was expanded and transfected with the Hoxb-9 targeting vectorcontaining the complementary hprtΔ5′ cassette. The minigene fragmentswere oriented in the targeting vectors so that, after targeting, theywould be in the correct order and orientation with the positiveselection cassettes (neo and puro) located between the loxP sites (FIG.2C). Double-targeted clones were identified (FIG. 2C), and half of theseclones would be expected to have both targeted alleles on the samechromosome (cis) and half should have the targeted alleles on differenthomologues (trans).

[0077] To induce the recombination between the loxP sites, severalindependent double-targeted (Hoxb-1 and Hoxb-9) clones were expanded andtransiently transfected with a Cre expression cassette and placed underHAT selection. Control transfections without Cre did not yield anyHAT-resistant clones. What follows is a more detailed description of themethod employed in this example.

[0078] As depicted in FIG. 1A, the PGKHprt minigene was modified by theinsertion of a loxP site from pBS64 (HindIII-EcoRI, Klenow blunt) intothe unique XbaI site (Klenow blunt) in the hprt intron. Insertion of theloxP site did not disrupt the cassette's HAT resistance function (notshown). The loxP-Hprt cassette was divided into two overlapping pieces:hprtΔ3′, which contains the PGK promoter, the hprt exons 1 and 2, theloxP-intron, hprt exons 3-6, and the SV40 poly A signal. In FIG. 1A,hprtΔ5′ and hprtΔ3′ have a 2 kb overlap including the loxP site;independently, hprtΔ5′ and hprtΔ3′ do not provide HAT resistance, butthey do when co-electroporated. HprtΔ5′ and hprtΔ3′ were ligated topositively selectable cassettes (hprtΔ3′ to the pol II neo gene andhprtΔ5′ to a PGK-puromycin resistance gene); in both cases, the positivemarkers replaced the deleted part of the loxP-Hprt cassette, ensuringthat, upon recombination, they are separated from the reconstitutedcassette. FIG. 1C depicts the general strategy for making deletionsconsisting of 3 steps: Step 1: conventional replacement-type genetargeting used to replace the Hoxb-1 gene with the hprtΔ3′-neo cassette;Step 2: ES cells identified as correctly targeted are used as asubstrate to insert the hprtΔ5′-puro cassette into the endogenous Hoxb-9gene by conventional replacement-style targeting (the cis configurationis illustrated here); and Step 3: transient expression of Cre inducesrecombination between the loxP sites which reconstructs a functionalhprt minigene. In FIG. 1 C4, the intra-chromosomal recombination pathwayis illustrated, and in FIG. 1 C5, cells with the recombinant (deleted)chromosome to be positively selected in HAT media and a chromosomal ringare generated by the intra-chromosomal pathway. This is believed to beunstable and lost during the growth of the colony.

[0079] The targeting vector for Hoxb-1 consists of a 3.5 kb BgIII-NcoIfragment (5′ homologous arm); the Hoxb-1 coding sequence (1.7 kbNco-BgIII) was replaced by the hprtΔ3′-neo cassette and a 2 kbBgIII-PvuII fragment (3′ homologous arm). The vector was linearized withSalI and a 10 μg was electroporated into hprt-negative AB2.2 ES cells.g418 selection was applied 24 hours after the electroporation andresistant clones were arrayed in 96 well plates, and targeted cloneswere detected by Southern analysis. A single targeted clone out of 384clones analyzed was identified with the predicted structure of thetargeted allele using probes 5′ and 3′ of the Hoxb-1 gene. This clonewas expanded and transfected with the Hoxb-9 targeting vector. Thetargeting vector for Hoxb-9 consisted of a 6.2 kb HindIII fragment ofhomology which included exon 1. The hprtΔ5′-puro cassette was clonedinto the unique SalI site in exon 1. The orientation of the cassette wassuch that, when targeted, the loxP sites in the hprtΔ cassettes would bein the same orientation. The vector was linearized and 10 μg of vectorwas electroporated into clone #298 AB2.2 cells and plated on SNLP (puroresistant SNL76/7 cells). Puromycin selection (5 μgml) was applied 24hours after electroporation. Resistant clones were arrayed in 96 wellplates and screened for targeted clones by Southern analysis.Double-targeted clones were detected at a frequency of 6%. Multipleindependent double-targeted clones were expanded and independentlytransiently transfected (by electroporation) with 20 mg of a supercoiledCre expression cassette pOG231. HAT selection was applied 48 hours afterthe electroporation. HAT-resistant clones were arrayed and analyzed bySouthern Blot analysis.

[0080] As evidenced by Table 1, two classes of double-targeted clonescould be distinguished by this assay. TABLE 1 Frequency (10⁻⁷) DeletionInterval Distance I II Inversion Hoxb9 - Hoxb1 90 kb 153 0.5 ND Hoxb9 -E2DH 3-4 cM 6 1 43 Gastrin - E2DH 1 Mb 470 1.8 334 E2DH - Wnt3 3-4 cM 304 19

[0081] Table 1 reports the frequency of Cre-mediated recombination as afunction of distance between the loxP sites. Deletion frequencies areillustrated for both Class I and Class II clones while inversionfrequencies are only illustrated for Class I clones.

[0082] One class (Type I) yielded HAT-resistant recombinants atfrequencies averaging 1×10⁻⁵ per treated cell, while a second class(Type II) yielded HAT-resistant clones at a much lower frequency.

[0083] Table 2 shows the frequency of Cre-mediated recombination as afunction of distance between the loxP sites. TABLE 2 Example BRECOMBINATION FREQUENCY ON THE MOUSE CHROMOSOME 11 Deletion InversionDuplication Interval Cis Trans Cis Trans Cis Trans Gastrin-E₂DH (1Megabase) 476 1 355 0 166 2 E₂DH-D11MIT199 (2 cM) 102 3 293 0 N/A N/AHoxB-E₂DH (3-4 cM) 2 0 43 0 N/A N/A E₂DH-Wnt3 (3-4 cM) 36 4 19 0 N/A N/AE₂DH-D11MIT69 (22 cM) 0 0 3 0 N/A N/A

[0084] Table 2 is similar to Table 1, except that the former shows anadditional deletion frequency for an additional interval(E₂DH-D11Mit69); it also shows duplication frequency; and finally itshows additionally deletion, inversion, and duplication frequencies forboth cis- and trans-.

EXAMPLE C Cis and Trans Recombination

[0085] Two types of clones might correspond to the cis or transconfiguration of the loxP sites. The HAT-resistant clones derived at ahigh frequency from Type I clones might be products in intrachromosomalrecombination or sister chromatid exchange (loxP sites in cis). Type IIclones might require interchromosomal or inter-sister-chromatidrecombination between homologous chromosomes (loxP sites in trans),which may occur relatively infrequently. These different pathways weredistinguished by analyzing the markers in recombinant HAT-resistantclones.

[0086] Deletion of the HoxB cluster from a chromosome double targeted incis would be accompanied by the loss of the neo and puro cassettes.These are either segregated (sister-chromatid pathway) or a ring isformed which is presumed to be unstable (FIG. 1). The loss of bothmarkers could be documented in most of the HAT-resistant clones derivedfrom Type I double-targeted clones, consistent with the hypothesized cisconfiguration of the Type I clones.

[0087] As anticipated, the consecutive targeting events and theCre-induced recombination event results in the formation of novelrestriction fragments (FIG. 2). External probes identify the noveljunction fragments using an NheI digest since there is not an NheI sitepresent in the loxP-hprt cassette. An internal probe confirms the lossof 90 kb of sequence by dosage difference between the wildtype and thedeletion clones (FIGS. 2 E and F).

EXAMPLE D E2DH-Gastrin 1 Mb Deletion

[0088] One of the goals behind the methods of the present invention isto construct chromosomal deletions so that regions of the genome can betested for tumor suppressor activity. Many candidate regions have beenidentified from loss of heterozygosity (LOH) studies. One well-definedregion in human breast cancer maps close to the Gastrin locus on humanchromosome 17 q close to BRCA1. Miki, et al., Science 266:66-71 (1994).This putative sporadic tumor suppressor locus maps in a conservedlinkage group on mouse chromosome 11 between Gastrin and the E2DH locus.The generation of hemizygous mice with a deficiency that encompassesthis locus functionally tests if this region contains a sporadic breastcancer gene that is involved in mammary neoplasia.

[0089] The large size of the regions which contain putative sporadictumor suppressor loci complicates substantially the use of deletionstrategy. In the absence of a YAC cloning contig which spans therelevant genetic interval, the gene order and orientations were notknown. This is an important consideration since both the order and theorientation of the Hprt minigene fragments will determine the type ofchromosomal rearrangement that is required to reconstruct a functionalHprt cassette. The possible orientations are illustrated in FIG. 1D. Therecombinant chromosomes include deletions, duplications, inversions, anddi- and acentric chromosomes (FIG. 1E). These rearranged chromosomes canbe distinguished on Southern blots by the appearance of novel junctionfragments, but the most rapid identification of the clones withdeletions can be obtained from selection using neomycin and puromycinresistance cassettes which have been configured to lie between the loxPsites in the to-be-deleted interval (see FIG. 1).

[0090] To construct ES cell lines with large deletions between theGastrin and the E2DH locus (containing SBC I) in the absence of a prioriknowledge of the gene order and orientation, all four possiblearrangements of the hprt minigene fragments were made and tested, onlyone of which will generate deletions. Two targeting vectors wereconstructed for each deletion endpoint representing the two possibleorientations of the hprt minigenes. The hprtΔ3′ cassette was targeted tothe E2DH locus with the alternative minigene orientations (A or B).Targeted clones representing both the A and B orientations were, inturn, transfected with the targeting vectors representing the differentorientations of the hprtΔ5′ cassette (A or B) at the Gastrin locus.Multiple independent, targeted clones were isolated representing thefour different minigene configurations to ensure that clones with thecis and the trans configurations were likely to be represented. Each ofthese clones was expanded, transfected with a Cre expression cassetteand plated under HAT selection. What follows is a more detaileddescription of the method employed in this example.

[0091] Overlapping λ phage containing the mouse E2DH locus were isolatedfrom a mouse 129Sv/Ev genomic library using a human E2DH cDNA probe.Unlike the duplicated human E2DH locus, the mouse locus is present at asingle copy. λ phage containing the mouse Gastrin locus were isolatedfrom the same library using a PCR fragment from the rat Gastrin cDNA.The mouse E2DH gene and Gastrin genes had not been mapped; to confirmthat these genes mapped to mouse chromosome 11, a hamster/mouse hybridcell line in which the mouse chromosome 11 is the only mouse geneticmaterial was hybridized to probes specific for the mouse Gastrin andE2DH loci.

[0092] Standard gene replacement targeting vectors were constructed fromthese genomic clones. E2DH vector: a total of 8.0 kb of homology wasused. The XhoI-XbaI 5.5 kb fragment containing the entire E2DH codingsequence was replaced with the hprtΔ3′ minigene cassette in bothorientations. In the Gastrin vector, a total of 7.5 kb of homology wasused. The 3.5 kb XhoI-NheI fragment containing the Gastrin coding regionwas replaced with the hprtΔ5′ cassette in both orientations.

[0093] The two vectors were separately transfected into AB2.2 ES cells.G418 resistant clones were obtained for each vector. Clones were griddedonto 96 well plates and screened for targeted clones. Targeted cloneswere identified at a ratio of 1/25 for the A orientation vector and 1/25for the B orientation vector. The two types of targeted ES cells wereassayed for totipotency by generating chimeras which tested for germline transmission. Totipotent E2DH-targeted ES cell lines wereidentified for both the A and the B orientation and these weretransfected with the vectors which target the Gastrin locus. Puromycinresistant clones were arrayed on 96 well plates and screened fortargeted clones. All four classes of double targeted clones wereobtained. For simplicity, this figure only shows the double targeted EScell having the hprtΔ5′ and hprtΔ3′ cassettes in the A orientation andin cis.

[0094] The double targeted ES cell clones were transfected with the Creexpression plasmid as previously described. HAT^(r) colonies wererecovered and sibselected to test for puromycin and G418 resistance.Individual clones were expanded and analyzed for junction fragmentsusing multiple probes. Each blot was hybridized with two probes, onefrom the E2DH locus and the other from the Gastrin locus. The frequencyof obtaining HAT resistant colonies from the different clones issummarized in Table 3. TABLE 3 Clones m HAT^(r) Category Class tested(10⁷) G418 Puro AA I 4 470 S S II 2 1 R R/S AB I 5 344 R R II 1 0 — — BAI 3 377 R R II 2 0 — — BB I 3 166 R R II 6 18 R/S R

[0095] Table 3 reveals the frequency of the Cre-mediated recombinationand retention of the markers in recombinant clones. All of the data isderived from the E2DH-Gastrin double targeted clones. The categories ofclones are illustrated in FIG. 1D, and the expected products aredescribed in FIG. 1E. Class I double-targeted clones give a highfrequency of HAT-resistant recombinants, while Class II clones give alow frequency of HAT-resistant clones. Retrospective analysis hasrevealed that the class I clones and Class II clones have the targetedgenes in cis and trans. S and R refer to resistance or sensitivity toG418 or puromycin as assayed by selection. Both resistant and sensitiveclones were recovered.

[0096] HAT-resistant clones were recovered from each of the fouralternative split minigene configurations. The individual clones withina specific orientation group could be placed into one of two classes,based on the frequency with which HAT-resistant clones could berecovered (Table 3). Selection analysis identified the AA class IHAT-resistant clones as those that had lost the neomycin and puromycinresistance genes; these clones are the most likely to have the desireddeletion. Since the AA class I clones gave the deletion product, thisallows predictions to be made on the likely products of the alternativeconfigurations: BB gives duplications, and AB or BA should giveinversions. These predictions have been confirmed by detailed molecularanalysis summarized in FIG. 3. In particular, the juxtaposition of thehprt minigene fragments which were previously positioned approximately 1Mb apart in the genome results in unique junction fragments that arespecific for the different types of rearrangement.

[0097] The AA type II clones yield HAT-resistant recombinants at a lowfrequency. It was hypothesized that these clones represented the caseswhere the deletion selection cassettes had integrated in trans.Interchromosomal recombination would result in both the neo and puroresistance genes being located on one chromosome, while thereconstructed hprt minigene would be on the homologue. Thus it would beanticipated that all of the positive selection markers would be retainedin such a cell. Sibselection identified two classes of HAT-resistantclones which were represented at approximately equal frequencies. Onetype only retained the neo cassette, and a second type retained both theneo and the puromycin resistance cassettes (Table 1A). The segregationof the puro resistance gene from the neo cassette is explained readilyif Cre-induced recombination between sister chromatids (FIG. 4)occurred. This occurrence was confirmed by the molecular analysis ofthese clones. While the clones with the duplicated and deletedchromosomes can be generated by either interchromosomal or non-sisterchromatid exchange, the clones which only carry the neo cassette canonly have arisen by the non-sister chromatid recombination pathway.These clones have been confirmed to carry both the deletion chromosomeand the non-recombinant chromosome with only the E2DH targeted locus(FIG. 4).

[0098] The clones with the deletion on one chromosome and theduplication on the other are genetically balanced. Therefor these cloneswere considered to be the best candidates for germ line transmission.Four independent clones were injected into blastocysts, representingclones descended from both the A and B orientation of the E2DH targetedallele. Alleles from three of these clones were transmitted into thegerm line, despite three cycles of subcloning and expansion. Segregationof the deletion and duplication alleles from a chimeric male isillustrated by gene dosage analysis (FIG. 5). Mice which are hemizygousfor this deletion (1 copy) are fully viable. Mice which are heterozygousfor the duplication (3 copies) or homozygous (4 copies) are also fullyviable (FIG. 5) and fertile.

EXAMPLE E Deletion of Two 34 Centimorgan Regions on Chronosome 11

[0099] Given the apparent insensitivity of the Cre-induced recombinationto the distance between the loxP-hprt substrates, two additionalexperiments were performed to investigate if Cre could delete a largerfragment. Two 3-4 cM intervals were chosen, proximal or distal to theE2DH locus on mouse chromosome 11. This region is syntectic with aregion on human chromosome 17q where loss of heterozygosity studies haveidentified several distinct regions that are likely to contain tumorsuppressor genes which are mutated in 30-70% of sporadic breast cancer.These regions have been termed SBCI, SBCII and SBCII (FIG. 6A).

[0100] Since the E2DH-Gastrin deletion had revealed the orientation ofthe E2DH locus, one of the A orientation E2DH-targeted clones wasselected for the proximal deletion and a B orientation E2DH-targetedclone was selected for the distal deletion. Targeting vectors (twoorientations) were constructed for the HoxB locus (Hoxb-9) and for Wnt3(see Roelink, et al., PNAS USA 87:4519-23 (1990)). Double-targetedclones were generated, transfected with the Cre expression cassette andHAT-resistant clones were selected. One vector orientation yielded G418and puro sensitive clones which were hypothesized to have a 3-4 cMdeletion, while the other orientation yielded HAT-resistant clones whichall retain the neo and puro cassettes. This latter category of cloneswere confirmed to be inversions of the 3-4 cM interval by molecularanalysis. The molecular analysis of the G418 and puro sensitive clonesidentified two classes of clones that occur with approximately equalfrequency. The first type of clone was consistent with a simple deletionevent, illustrated for the Hoxb9-E2DH deletion (FIG. 6D). The otherclass of clone exhibited the expected deletion junction fragment, butalso retained a junction fragment that is diagnostic for the targetedchromosome, but should have been lost during the deletion event(illustrated for the E2DH-Wnt3 deletion in FIG. 6D). The retention ofthis junction fragment and the acquisition of the expecteddeletion-specific fragment in about half the clones can be explained bytwo different recombination pathways. The pure clones are believed to beproducts of an inter-chromosomal pathway (FIG. 1C), while those clonesthat retain the primary targeted allele may reflect a sister-chromatidexchange. While the duplicated chromosome should be segregated to adaughter cell and does not carry the reconstructed hprt minigene,extensive metabolic co-operation between the hprt⁺ and hprt³¹ ES cellsin a colony facilitate cross rescue and substantial contribution of thehprt³¹ daughter cells to the HAT-resistant clones. What follows is amore detailed description of the method employed in this example.

[0101] E2DH and Hoxb9 vectors have been described previously. Wnt3genomic clones were isolated from a 129Sv/Ev genomic library using acDNA probe. Conventional replacement targeting vectors containing 7.0 kbof homology were constructed. The hprtΔ5′ cassette replaces a 2.1fragment (contains exons 3 and 4) of the Wnt3 gene. The Hoxb9 vectorsand the Wnt3 vectors were independently targeted into the E2DH targetedcell lines. An “A” orientation clone was used for the Hoxb9 targetingvectors. Transfected cells were selected in puromycin and targetedclones were identified as previously described. Multiple independentdouble targeted clones were transfected with the Cre recombinase.HAT-selection was used to isolate recombinant clones which weresib-selected and tested by Southern analysis for the predicted junctionfragments.

EXAMPLE F Using a Virus Rather than Targeting to Effect theRecombination

[0102] Rather than relying on traditional targeting techniques, eitheror both of the desired deletion endpoints can be added to the genome bymeans of a retrovirus. In this example, a viral vector is used to insertthe endpoint at the 5′ end only. FIG. 7 is a schematic representation ofa provirus structure suitable for this use, which is comprised of anhprtΔ5′ minicassette, a loxP site and a puromycin resistance gene.Hence, the provirus structure is similar to the non-viral targetingvectors described in the previous examples.

[0103] In this example, only the 5′ endpoint was inserted using theviral vector depicted in FIG. 7, though in other embodiments, bothendpoints can be added using viral vectors. Chromosomal deletions wereinduced using the methods substantially as described in the previousexamples. Hence, insertions are made at the two endpoints framing thedesired chromosomal deletion. The insertions are preferably made one ata time, and involve replacing a first native sequence (i.e., the firstendpoint) on the chromosome of interest with a first selection cassette.This selection cassette consists of three elements: a first selectablemarker, a loxP site located in the hprt minigene intron, and a firstportion of a second selectable marker, preferably a non-functionalfragment of an Hprt minigene cassette. The first selectable marker ispreferably a neomycin resistance gene (hprtΔ5′ cassette) or a puromycinresistance cassette (hprtΔ3′ cassette). The cells expressing the marker(either the neomycin resistance gene or the puromycin resistance gene)are then selected. Next, the process is essentially repeated for secondendpoint on the chromosome of interest. Thus, the cells selected possessboth loxP sites framing the desired portion of the chromosome to bedeleted. The difference in this step is that the hprt minigenefragment—also non-functional—is the complementary portion to thatinserted into the first endpoint. Third, the selected cells arecontacted with Cre, which may be expressed in one of the three waysdescribed above, which induces recombination between the loxP sites.This recombination generates a fully functional Hprt minigene. Thisminigene provides resistance to HAT selection in hprt deficient cells.Additionally, the positive selectable markers are positioned so thatfollowing recombination, they are lost from the deleted chromosome.Therefore, the methods for inducing the deletion described in thisexample are nearly identical to those for practicing the method using anon-viral vector. Inherent differences in method and technique thatresult from using a viral versus non-viral vector are well-known to theskilled artisan, hence a detailed description of any embodiment of themethod of the present invention involving non-viral vectors could beeasily adapted by the skilled artisan using a viral vector. Therecombination efficiencies obtained from using the viral vector(described in FIG. 7) at the 5′ endpoint are shown below in Table 4.TABLE 4 # cell out of 10⁷ cre electroporated ES cells HAT^(r) Puro^(r)NO Drug Rec Efficiency plate #3 275 1.62 × 10⁵ 3.9 × 10⁵ 1.7 × 10⁻³plate #4 388 2.54 × 10⁵ 3.9 × 10⁵ 1.5 × 10⁻³ Average 331 2.09 × 10⁵ 3.9× 10⁵ 1.6 × 10⁻³

EXAMPLE G Frequency of Cre-induced Deletion Between E₂DH and D11 Mit199Using an Improved Targeting Vector

[0104] This example illustrates the enhanced Cre-induced deletionfrequency using a different targeting vector compared with that used inthe previous examples. The details of the protocol are substantially asdescribed in the previous examples. Details of this “improved vector”compared with the vector used in the previous examples are shown inFIGS. 10 through 12 (the original vector is shown by comparison in FIG.12). FIG. 10 shows a map of an exemplary 5′ endpoint targeting vectorautomatically excised out of a phage clone isolated from the 5′ anchorlibrary. The 5′ anchor library is shown in FIG. 9; this anchor librarycontains the expression cassettes 5′ hprt, neomycin resistance gene, andtyrosinase gene. FIG. 11 shows a map of an exemplary 3′ endpointtargeting vector automatically excised out of a phage clone isolatedfrom the 3′ anchor library. The 3′ anchor library is shown in FIG. 8;this anchor library contains the expression cassette 3′ hprt, puromycinresistance gene, and k14-agouti gene. FIG. 12 shows a map of pG12WT(Wildtype 3′ hprt cassette plasmid for making chromosomalrearrangements). At the bottom of FIG. 12, a comparison of the sequenceused to generate the data in Examples B-E versus the sequence used togenerate the data in this and following examples is shown. As evidencedby FIG. 12, the two sequences are identical to pG12 except that themutation in 3′ hprt of the wildtype cassette plasmid has been fixed.

[0105]FIG. 13 shows the portion of the mouse chromosome 11 at which thedeletion strategy is directed; FIG. 13 also shows the generalcomposition of the selection cassettes positioned at the chromosomeendpoints, and the position of the Cre-induced deletion interval,E_(2DH-D)11Mit199.

[0106] Table 4 shows the frequency of Cre-induced deletion between E₂DHand D11Mit199, which can be compared with Tables 1A, 1B, and 4. Thefrequency shown is the number of HAT-resistant colonies perCre-electroporated cell. The numbers are obtained by averaging data fromat least two experiments with at least two cell lines (except for thenew vector in the trans configuration). A comparison of the datapresented in Table 4 with those in Tables 1, 2, and 3 reveal that thecassette shown in FIG. 11 mediates recombination approximately 10³ moreefficiently than the cassette used to generate the data in Tables 1, 2,and 4.

EXAMPLE H Frequency of Cre-induced Deletion Between E₂DH and D11 Mit69Using an Improved Targeting Vector

[0107] Similar to Example H, this example also illustrates the enhancedCre-induced deletion frequency using a different targeting vectorcompared with that used in the previous examples. Example H illustratesthe Cre-induced deletion frequency between E₂DH and D11Mit199—a distanceof about 2 CM. By contrast, Example I illustrates the Cre-induceddeletion frequency between E₂DH and D11Mit69—a distance of about 22 CM).The details of the protocol are substantially as described in theprevious examples. Details of this “improved vector” compared with thevector used in the previous examples are shown in FIGS. 10 through 12(the original vector is shown by comparison in FIG. 12). FIG. 10 shows amap of an exemplary 5′ endpoint targeting vector automatically excisedout of a phage clone isolated from the 5′ anchor library. The 5′ anchorlibrary is shown in FIG. 9; this anchor library contains the expressioncassettes 5′ hprt, neomycin resistance gene, and tyrosinase gene. FIG.11 shows a map of pG12WT (Wildtype 3′ hprt cassette plasmid for makingchromosomal rearrangements). The sequence is identical to pG12 exceptthat the mutation in 3′ hprt has been fixed.

[0108]FIG. 14 shows the portion of the mouse chromosome 11 at which thedeletion strategy is directed; FIG. 14 also shows the generalcomposition of the selection cassettes positioned at the chromosomeendpoints, and the position of the Cre-induced deletion interval,E₂DH-D11Mit69. Table 6 shows the frequency of Cre-induced deletionbetween E₂DH and D11Mit69, which can be compared with Tables 1A, 1B, and4. TABLE 6 Example H Markers Frequency HAT^(r), Puro^(s), Neo² 5.8 ± 3.3× 10⁻⁶ (n = 5)/2 × 10⁻⁵ (n = 1) HAT^(r), Puro^(r), Neo^(r) 1.1 ± 0.4 ×10⁻⁵ (n = 10)/3 × 10⁻⁵ (n = 1)

[0109] Two frequencies are reported in each row, reflecting two separatetrials. The “frequency” of Cre-induced loxP recombination is expressedas the number of HAT^(r) colonies per Cre-electroporated cell. Thenumber of independent doubly targeted cell lines is denoted by “n=.”TABLE 5 Example G 3′ hprt cassette Cis Trans Old (mutant) 2 ± 0.5 × 10⁻⁵6.5 ± 3.2 × 10⁻⁷ New (wildtype) 2.3 ± 1.3 × 10⁻² 1.5 × 10⁻⁴

We claim:
 21. A method for deleting a selected region of geneticmaterial in mice comprising the steps of: inserting a first selectioncassette at a 5′ end of said selected region using conventional genetargeting methods, said first selection cassette comprising a firstselectable marker coding sequence, a first loxP recombination site, anda first portion of a second selectable marker coding sequence; selectingembryonic stem cells expressing said first selectable marker codingsequence; inserting a second selection cassette at a 3′ end of saidselected region using conventional gene targeting methods, said secondselection cassette comprising a third selectable marker coding sequence,a second loxP recombination site, and a remaining portion of said secondselectable marker coding sequence; selecting embryonic stem cellsexpressing said third selectable marker coding sequence; expressing Crerecombinase to produce recombination between said first and second loxPsites; selecting embryonic stem cells expressing said second selectablemarker coding sequence, wherein the selected region of genetic materialis deleted; injecting selected embryonic stem cells expressing saidsecond marker into a recipient blastocyst; and implanting saidblastocyst into a foster mother.
 22. The method of claim 21 wherein saidfirst selectable marker coding sequence is a puromycin resistance gene,said second selectable marker coding sequence is an Hprt gene, and saidthird selectable marker coding sequence is a neomycin resistance gene.23. A method for creating inversions of a selected region of geneticmaterial in mice comprising the steps of: inserting a first selectioncassette at a 5′ end of said selected region using conventional genetargeting methods, said first selection cassette comprising a firstselectable marker coding sequence, a first loxP recombination site, anda first portion of a second selectable marker coding sequence; selectingembryonic stem cells expressing said first selectable marker codingsequence; inserting a second selection cassette at a 3′ end of saidselected region using conventional gene targeting methods, said secondselection cassette comprising a third selectable marker coding sequence,a second loxP recombination site, and a remaining portion of said secondselectable marker coding sequence; selecting embryonic stem cellsexpressing said third selectable marker coding sequence; expressing Crerecombinase to produce recombination between said first and second loxPsites; selecting embryonic stem cells expressing said second selectablemarker coding sequence, wherein the selected region of genetic materialis inverted; injecting selected embryonic stem cells expressing saidsecond marker into a recipient blastocyst; and implanting saidblastocyst into a foster mother.
 24. A method for creating a definedchromosomal deficiency, deletion, inversion or duplication in micecomprising the steps of: identifying a desired region of a chromosome ofinterest to be targeted; inserting two native sequences at each endpointof said region of said chromosome of interest using a first and a secondtargeting vector, each comprised of one or more selectable marker codingsequences and a loxP site and an hprt fragment coding sequence;transiently expressing Cre recombinase to produce recombination betweeneach of two said loxP sites; whereby upon chromosomal rearrangementinduced by said Cre recombinase, a functional Hprt expression cassetteis reconstructed; selecting embryonic stem cells expressing Hprt,wherein the region of said chromosome of interest is deficient, deleted,inverted, or duplicated; injecting selected embryonic stem cellsexpressing Hprt into a recipient blastocyst; and implanting saidblastocyst into a foster mother.
 25. A transgenic mouse produced by themethod of claim 23.